The present invention is in the field of molecular biology; more particularly, the present invention describes nucleic acid sequences and amino acid sequences for mammalian EDG-7 receptor homologs, and particularly for human EDG-7 receptor homologs.
The family of edg receptors are commonly grouped with orphan receptors because their endogenous ligands are not known (for example see Hla T and Maciag T (1990) J Biol. Chem. 265:9308-13; U.S. Pat. No. 5,585,476). Recently, however, lysophospatidic acid has been demonstrated to be the endogenous ligand for the edg-2 receptor (Hecht et al. (1996) J. Cell. Biol. 135: 1071-1083; An et al. (1997) Biochem. Biophys. Res. Comm. 213: 619-622).
The edg family of receptors is seven transmembrane G protein coupled receptors (T7Gs or GPCRs). T7Gs are so named because of their seven hydrophobic domains that span the plasma membrane and form a bundle of antiparallel xcex1 helices. These transmembrane segments (TMS) are designated by roman numerals I-VII and account for structural and functional features of the receptor. In most cases, the bundle of helices forms a binding pocket; however, when the binding site must accommodate more bulky molecules, the extracellular N-terminal segment or one or more of the three extracellular loops participate in binding and in subsequent induction of conformational change in intracellular portions of the receptor. Specifically: the TM-VII is generally a highly conserved portion of the T7G receptors, and is often critically involved in ligand binding and receptor activation; the intracellular carboxy-terminal is involved in interactions with intracellular proteins, including those that transduce intracellular signals upon receptor activations; the carboxy-terminal is usually hydrophilic and highly antigenic relative to the receptor polypeptide as a whole and shows greatly reduced conservation.
Once the receptor is activated, the receptor, in turn, interacts with an intracellular G-protein complex which mediates further intracellular signaling activities, including: generally, the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate; activation of protein kinases; and alteration in the expression of specific genes.
T7G receptors are expressed and activated during numerous developmental and disease processes. Identification of a novel T7G receptor provides the opportunity to diagnose or intervene in such processes, and the receptor can he used in screening assays to identify physiological or pharmaceutical molecules which trigger, prolong or inhibit its activity or differentially modulate distinct intracellular pathways which are controlled from T7G receptors.
The invention provides isolated and unique nucleotide sequences that encode novel mammalian EDG-7 receptor homologs, and particularly, novel human EDG-7 (HEDG-7) receptor homologs. Herein, the nucleotide sequence encoding HEDG-7 is designated hedg-7.
The present invention also relates to the isolated and unique nucleotide sequences of the complement of hedg-7 mRNA. In addition, the invention features nucleotide sequences, which hybridize under stringent conditions to hedg-7.
The present invention also relates to nucleotide sequences that encode fragments or portions of hedg-7, or complements thereof, in addition to expression vectors and host cells comprising such nucleotide sequences.
The present invention also provides amino acid fragments, particularly fragments in the TM-VII and carboxy-terminal domains that are useful as antibodies for HEDG-7.
Furthermore, the invention relates to the use of the nucleotide sequences of hedg-7 and the amino acid sequences of HEDG-7, or its variants, in the diagnosis or treatment of diseased cells and/or tissues associated with aberrant expression of hedg-7.
Additional aspects of the invention include the antisense DNA of hedg-7; cloning or expression vectors containing hedg-7; host cells or organisms transformed with expression vectors containing hedg-7; chromosomal localization of hedg-7; expression and tissue distribution of hedg-7; a method for the production and recovery of purified HEDG-7 from host cells; purified protein, HEDG-7, which can be used to identify inhibitors for the downregulation of signal transduction involving HEDG-7; and methods of screening for ligands of hedg-7 using transformed cells.
In particular, the present invention provides an isolated nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence comprising nucleotides 16-1170 of FIG. 1A;
(b) the nucleotide sequence comprising nucleotides 13-1167 of FIG. 1B;
(c) a nucleotide sequence with 70% sequence identity to (a) or (b), more preferably at least about 80-85% sequence identity, and even more preferably at least about 90% sequence identity, and most preferably at least about 95% sequence identity, and which nucleotide sequence hybridizes under stringent conditions to the nucleotide sequence of (a) or (b), respectively, or portions thereof;
(d) a nucleotide sequence which encodes the amino acid sequence of FIG. 2A; and
(e) a nucleotide sequence which encodes the amino acid sequence of FIG. 2B.
There is also provided: expression vectors; host cells; purified amino acid sequences; complementary nucleic acid sequences; biologically active fragments; and hybridization probes, for such nucleotide sequences and their encoded amino acid sequences.